The present invention relates to antibodies land other binding molecules specific for hepatitis B viral antigens (HBV), peptides comprising epitopes recognised by such molecules, and cell lines capable of producing antibodies. The invention is further concerned with the use of such molecules in diagnosis of hepatitis B virus (HBV).
The virus that causes hepatitis B or serum hepatitis appears to infect only man and chimpanzees. Hepatitis B virus (HBV) infection in humans is widespread.
The hepatitis infection is transmitted by three general mechanisms: (1) by parenteral inoculation of infected blood or body fluids, either in large amounts as in blood transfusions or in minute amounts as through an accidental skin prick; (2) by close family or sexual contact; and (3) by some mothers, who infected during pregnancy, transmit the virus to their new-born children. Under natural conditions, HBV is not highly contagious. Transmission by inhalation occurs rarely, if ever.
The transmission route through contaminated blood or blood products is a major threat to the human health.
Infection with HBV often results in subclinical or acute self-limited liver disease or can result in chronic long-term infection. Chronic HBV infection elicits a spectrum of disease entities ranging from the most severe form of chronic active hepatitis (CAH) to less severe chronic persistent hepatitis (CPH) to the asymptomatic carrier (ASC) state. An array of diagnostic assays have recently been developed to aid the clinician in differentiating hepatitis B virus infections from other forms of viral hepatitis (i.e., HAV, HEV, HCV). However, the ability to distinguish between an acute hepatitis B (AH-B) infection and symptomatic chronic hepatitis B (CH-B) infection is still problematic. This is especially true since CAH and CPH patients often demonstrate a cyclic pattern of hepatitis characterised by acute exacerbations (A.E.) of liver injury alternating with normal liver function.
After infection with HBV, large quantities of the virus and associated particles are present in the serum. During symptomatic phases of infection, both acute and chronic HBV patients have elevated liver enzyme levels, possess the hepatitis B surface antigen (HBsAg) in their serum, and produce antibodies to the nucleocapsid antigen (HBcAg). Antibodies specific for the HBsAg or the hepatitis B e antigen (HBeAg) are not detected. The appearance of antibody to HBsAg is usually not observed until approximately two months following disappearance, of circulating HBsAg. The viral particles present in the serum are known to shed their surface coat exposing the nucleocapsid, known as the core antigen (HBcAg). Antibody production of HBcAg occurs early in the course of the acute phase of HBV infection and can persist for many years, and chronically infected patients can produce high titers of anti-HBc antibodies.
The HBsAg is established as the most important marker of acute or chronic hepatitis B infection, detectable in serum of infected individuals. HBsAg screening of donor blood for example, is essential to avoid transmission of hepatitis B. It is clear that sensitivity is of utmost importance in diagnostic HBV assays.
HBV Surface Antigens (IBsAg)
The HBV surface antigens (HBsAg) are the translational products of a large open reading frame (ORF) that is demarcated into three domains; each of these domains begins with an in-frame ATG codon that is capable of functioning as a translational initiation site. These domains are referred to as Pre-S1, Pre-S2, and S in their respective 5xe2x80x2 to 3xe2x80x2 order in the gene. Thus, these domains define three polypeptides referred to as S or HBsAg (226 amino acids), Pre-S2+S (281 amino acids), and Pre-S1+Pre-S2+S (289-400 amino acids), also referred to, respectively, as major protein (S-protein), middle protein (M-protein), and large protein (L-protein) (Toillais et al., 1985, Nature, 317, 489-495).
Definition of an HBsAg Subtype
The HBsAg in the viral envelope part of HBV has one well-characterised group specific determinant xe2x80x9caxe2x80x9d and two sets of mutually exclusive subtype determinants d/y and w/r. Thus four major subtypes of HBsAgxe2x80x94adw, ayw; adr, and ayrxe2x80x94denote the phenotypes of the virion (Le Bouvier et al., 1975, Amer. J. Med. Sci. 270, 165). Subdivision of xe2x80x9caxe2x80x9d specificity into al, a2, a3, and other intermediate specificities which are later redefined as subdeterminants of w (w1-w4) at an international workshop in Paris in 1975 (Courouce et al., 1976, BibI Hematol, Basel, Karger, vol. 42), the issue of HBsAg subtypes acquired a considerable degree of complexity. These subtypes were ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, and adr. With the identification of the q determinant (Magnius et al., 1975, Acta Pathol Micr Scand, 83B, 295-297) the number of subtypes increased from eight to nine, due to the subdivision of the adr subtype into a q-positive and a q-negative category (Courouce-Pauty et al., 1978, Vox Sang, 35, 304-308). Sequencing of complete genomes encoding adw2 and ayw3 subtypes revealed numerous substitutions throughout the genome (Valenzuela et al., 1980, ICN-UCLA, Symposia on Animal Virus Genetics, NY, Ac. Press, pp57-70). A number of these substitutions in the S-gene were claimed to be associated with the expression of d and y specificity (Okamoto et al., 1986, J Gen Virol, 67, 2305-2314). Analysis of reactivity patterns with monoclonal antibodies after chemical modification of HBsAg revealed the importance of Lys 122 for the expression of the d determinant (Peterson et al., 1984, J Immun, 132, 920-927). Later studies on two blood donors carrying surface antigens of compound subtypes adyr and adwr respectively, showed that amino acid substitutions at position 122 and 160 alone explained the expression of d/y and w/r specificity, respectively (Okamoto et al., 1987, J Virol, 61, 3030-3034). Both the d to y and w to r changes were mediated by a shift from Lys to Arg at the corresponding positions. Therefore, major subtypic variations of HBsAg exist.
Definition of an HBsAg Genotype
Sequencing of viral genomes, and comparison has defined four genomic groups of HBV on a divergence of 8% or more of the complete genome, and were designated with A-D (Okamoto et al., 1988, J Gen Virol, 69, 2575-2583). Genomes encoding the subtype adw were found in genomic groups A-C, while the genomes encoding ayw were all found in group D and group B (Sastrosoewignjo et al., 1991, J Gastroenterol Hepatol, 6, 491-498). Genomes encoding both the adr and ayr subtype occurred in genomic group C alongside with adw.
Also two new genotypes of HBV designated with E and F were recently identified (Norder et al., 1994, Virology, 198, 489-503).
Immune Escape Mutants in Relation to Genotypes
Apart from the genetic variability of HBV based on the divergence of HBV strains over long periods of time resulting in geographically related subtypes and genotypes, considerable interest has recently also been focused on two kinds of immune escape mutants. The first of these to be described was a mutation from Trp 28 to a stop codon in the precore sequence, that specifically prevented the expression of HBeAg although leaving that of HBcAg unaffected (Carman et al., 1989, Lancet, ii, 588-591; Brunetto et al., 1991, Proc Natl Acad Sci USA, 88, 4186-4190).
Vaccine escape mutants are described involving the xe2x80x9caxe2x80x9d determinant of HBsAg, an important part of which is formed by a loop encompassing amino acid residues 139-147 stabilised by a disulphide bridge between two cysteinic residues at these positions (Waters et al., 1991, Virua Res, 22, 1-12; Stirk et al., 1992, Intervirology, 33, 148-158). One mutation from Gly to Arg at residue 145 of HBsAg was revealed in several vaccinees in Italy (Carman et al., 1990, Lancet, ii, 325-329) and Singapore (Harrison et al., 1991, J Hepatol 13 (suppl 4), S105-107). Another presumed vaccine escape mutation from Lys to Glu at position 141 has only been reported from West Africa (Allison et al., 1993, Abstr. Ixth Int Congr of Virology, Glasgow, pp1-118; Howard et al, 1993, Abstr. Int Symp on Viral Hepatitis and Liver Disease, Tokyo, pp1-75). Interestingly, this mutant has so far only been found in association with the ayw4 subtype. More recently various new mutants were among others described in literature by Brind et al., 1997, J. of Hepatology 26: 228-235; Kohno et al., 1996, J.of gen. virol. 77: 1825-1831; Ni et al., 1995, Res. Virol. 146: 397-407.
From the above it is clear that all sorts of mutations in the HBsAg proteins have to be detected in order to screen blood from donors and other sources.
For example Okomoto et al. (1992) already showed that the affinity between HBsAg mutants and monoclonal anti-HBs antibodies with known epitope specificity was impaired by substitution at amino acid 145 or 126 in the S-region (Okamoto et al., 1992, Pediatric Research 32: 264-268). Substitution of arginine for glycine at amino acid 145 in the yeast vaccine also results in markedly reduced binding by monoclonal antibodies (Waters et al., 1992, J. of clin. invest. 90: 2543-2547). It is further described that the antigenicity of HBsAg is weakened by substitution of amino acid 141 (Earthigesu et al., 1994, J. Gen. Virol. 75: 443-448).
Already various cases of HBsAg positivity which have been missed because of failure of current serological assays to detect some variant forms of the antigen have been described (Suzuki et al., 1995, Int. Hepatology Comm. 4: 121-125; Carman et al., 1995, The Lancet 345: 1406-1407; Jongerius et al., 1997, Ned Tijdschr Geneesk 141 (22) 1128).
Facing the above problems, many diagnostic companies changed their assays by using polyclonal antibodies as capture antibodies instead of using highly specific monoclonal antibodies.
However, drawbacks of using polyclonal antibodies are mainly directed to poor reproducibility, and unknown specificity and sensitivity of the individual antibodies in the total of all antibodies present in the polyclonal serum.
It will therefore always be unknown if newly documented variants will be detected before screening.
Another approach is finding new monoclonal antibodies to the S-region of HBsAg which recognise a well conserved region.
The S-region of HBsAg is of utmost importance and therefore the first region to investigate. The pre-S region of HBsAg is also a possible candidate although there are variants of HBsAg known who do not have a pre-S encoded protein in their virus material (Santantonio et al., 1992, Virology, 188, 948-952). In the same reference, the heterogeneity of HBV pre-S sequences coding for envelope proteins by DNA amplification and direct sequencing of viral genomes is described. In some patients deletions in the pre-S region, mainly clustered at the amino terminal end of the pre-S2 region, were found. The data indicated a high prevalence of HBV genomes that can only express deletion mutants of pre-S2 proteins and most of them cannot express a pre-S2 protein at all. According to the results most of the deletions should not prevent HBs synthesis.
The described heterogeneity of pre-S mutants predicts that false negative results may be obtained when enzyme immunoassays with monoclonal antibodies to pre-S proteins are used solely for their detection in sera of chronic carriers.
It is to the above problem the present invention is addressed.
According to the first aspect of the present invention, there is provided a molecule which is capable of specifically binding to a hepatitis B antigen determinant and which either is or cross-competes with a monoclonal antibody directed against at least part of the amino acid sequence:
RDSHPQAMQWNSTTFHQALLDPRVRGLYFPAGGSSSGT (SEQ ID NO: 1).
Preferred fragments comprise at least part of the amino acid sequence:
RDSHPQAMQWNSTTFHQAL (SEQ ID NO: 2), or
SHPQAMQWNSTTFHQALLDPR (SEQ ID NO: 3), or
ALLDPRVRGLYFPAGGSSSGT (SEQ ID NO: 4).
More preferred fragments are MQWN (SEQ ID NO: 5), STRFHQA (SEQ ID NO: 6), or VRGLYFPA (SEQ ID NO: 7), respectively.
A preferred molecule which is capable of specifically binding to a hepatitis B antigen determinant and which either is or cross-competes with a monoclonal antibody secreted by cell line HB.OT104A, HB.OT107C or HB.OT230B. These cell lines have been deposited at the European Collection of Animal Cell Cultures (ECACC), Centre for Applied Microbiology and Research, Salisbury, Wiltshire SP4 OJG, United Kingdom, under the accession numbers ECACC-97062610, ECACC-98042805 or ECACC-97062608, respectively.
Said hepatitis B antigen determinant is located at the pre-S region of HBV.
A specific binding molecule such as an antibody cross-competes with another if it binds to precisely the same, or a conformationally linked, location as the other. Conformationally linked locations may be adjacent locations on the polypeptide chain of the antigen or they may be linked by virtue of the secondary structure of the polypeptide chain, which can cause adjacent folding of otherwise non-adjacent regions. Cross-competition experiments are relatively easy to carry out (Waters et al., 1991). and so it is a straightforward matter to determine whether a given antibody or other specific binding molecule cross-competes with the monocional antibody specifically referred to above.
Specific binding molecules which at least partially cross-compete with the specified monoclonal antibodies (i.e. whose cross-competition is significantly greater than %) are useful in the invention. Specific binding molecules which totally cross-compete (i.e. whose cross-competition is not significantly less than 100%) are preferred, at least in some circumstances.
Specific binding molecules useful in the invention will often themselves be antibodies. While polyclonal antibodies are not excluded, monoclonal antibodies will generally be preferred because of their much more precise specificity. Monoclonal antibody technology has become well established since the original work by Kxc3x6hler and Milstein (1975, Nature, 256, 495) and there are today many available protocols for the routine generation of monoclonal antibodies. Suitable techniques, for example, are those of Gefter et al., (1977, Somatic Cell Genetics, 3, 231), Kxc3x6hler et al., (1976, Euro. J. Immuvirol., 292-295) and Goding (xe2x80x9cMonoclonal antibodies: Principle and Practicexe2x80x9d (2nd Edition, 1986) Academic Press, New York). Typically, the protocol used is as follows:
an experimental animal (such as a mouse) is immunologically challenged with the antigen against which antibodies are to be raised;
the spleen cells of the animal are then fused to cells of a myeloma cell line, and the resultant hybridoma fusion cells plated out on selective medium;
screening for specific antibodies is undertaken by any suitable technique, for example by the use of anti-immunoglobulin antibodies from another species.
While the use of human monoclonal antibodies may in principle be preferred for certain applications, particularly human therapy and in vivo diagnosis, technical difficulties render conventional hybridoma technology inappropriate for the generation of many human monoclonal antibodies. Non-human monoclonal antibodies, such as of murine origin, are therefore often used in practice.
Chimeric antibodies, particularly chimeric monoclonal antibodies, are also included within the scope of the invention. Such chimeric antibodies include sufficient amino acid sequences from HB.OT104A, HB.OT107C; HB.OT230B to have their characteristic specificity. At the minimum, the complementary determining regions of the specified antibody will be present to a sufficient degree to maintain specificity. It may be entire VH and VL domains will be present, or even entire antibody binding fragments such as the enzymatically derived Fab or F(abxe2x80x2)2 fragments.
Various different technologies exist for preparing chimeric antibodies. For example, chimeric antibodies consisting of a human C region fused to a rodent V region have been described (Morrison et al., 1984, PNAS, 81, 6851-6855; Boulianne et al., 1984, Nature, 312, 643-646; Neuberger et al., 1985, Nature, 314, 268-270).
Fully humanised antibodies, particularly monoclonal antibodies, are also within the scope of the invention. There are currently three separate methods for humanising non-human (particularly murine) antibodies. Reichmann et al. (!988, Nature, 332, 323-327) used site-directed mutagenesis on ssDNA. In another approach both Jones et al. (1986, Nature, 321, 522-525) and Queen et al. (1989, PNAS, 86, 10029-10033) constructed the whole V region using overlapping oligonucleotides incorporating the rodent complementarity-determining regions (CDRs) on a human framework. More recently, Lewis and Crowe (1991, Gene, 101, 297-302) have adapted polymerase chain reaction (PCR) methodology to graft rodent CDRs onto human immunoglobulin frameworks.
The amino acid sequences of the heavy and light chain variable domains of the monoclonal antibodies can be determined from cloned complementary DNA and the hypervariable regions (or complementarity determining regions CDRs) identified according to Kabat et al. (in xe2x80x9cSequences of Proteins of Immunological Interestxe2x80x9d, US Department of Health and Human Services, US Government Printing Office, 1987). Using any of the above methods these CDRs can be grafted into a human framework.
The single domain antibodies (dAbs) of Ward et al. (1989, Nature, 341, 544-546), represents another class of specific binding molecules (whether or not they are properly to be regarded. as xe2x80x9cantibodiesxe2x80x9d), which can be used in the scope of the present invention. In this approach, PCR or other appropriate technology is used to clone a VH or VL gene and express it in a heterologous host, such as E. coli. 
The heavy and light chain variable domains can be amplified from the hybridoma using the polymerase chain reaction (PCR) and cloned in expression vectors. The isolated variable domains can be screened for binding to antigen and their affinity determined. Other single domain antibodies can be obtained directly by amplifying by the rearranged variable domain genes from the spleen DNA of an immunised mouse. The amplified DNA can be cloned into a vector and then screened for antigen binding activity. A refinement using bacteriophage as an expression vector allows the phage carrying the variable genes to be selected directly with antigen because they are expressed on the cell surface (McCafferty et al., 1990, Nature, 348, 552-554).
The dabs technology indicates how recombinant DNA methodology is completely changing the generation of molecules having specific binding capabilities. For this reason if no other, the invention should not be regarded as being restricted to antibodies, as understood in the classical sense (whether polyclonal or monoclonal).
According to the second aspect of the present invention, there is provided a cell line or cell culture capable of expressing, and preferably secreting, specific binding molecules as described above.
Within this aspect of the invention are the hybridoma cell lines which have been specifically referred to above. These cell lines have been deposited at the European Collection of Animal Cell Cultures (ECACC), Centre for Applied Microbiology and Research, Salisbury, Wiltshire SP4 OJG, United Kingdom, under the accession numbers and dates shown in the following table:
These deposits have been made under the terms of the Budapest Treaty.
A generalised method for making other monoclonal antibodies has been described above. To ensure that they are within the scope of the invention, a simple cross competition experiment can be reacted with antibodies secreted by the deposited cell line.
Specific antibodies and other binding molecules in accordance with the invention are useful in diagnosis and in therapy.
The above defined antibodies and other binding molecules can be used in diagnostic applications in isolation or in combination with antibodies specifically directed to other regions of the HBV like the S-region.
If used in isolation, another test have to be performed in order to detect the HBV variants which lack the pre-S region.
If used in combination, which is preferred, only one test is necessary. In this instance, the antibodies directed against the S-region, do recognise also the HBV variants which do lack the pre-S region.
The antibodies and other binding molecules according to the present invention do overcome missing HBV infected individuals in the diagnosis of HBV infection.
With these antibodies, the HBsAg test-concept based on the S-region of HBV could be improved. Mutant detection of HBsAg is confirmed.
The antibodies and other binding molecules according to the present invention are useful tools for the detection of HBV expression in cells and cell extracts both in vivo and in vitro, for purification purposes and for a variety of biochemical and immunological analysis techniques to study the function of these proteins.
According to the third aspect of the present invention, there is provided a peptide comprising at least part of the amino acid sequence RDSHPQAMQWNSTTFHQALLDPRVRGLYFPAGGSSSGT (SEQ ID NO: 1).
A preferred embodiment is a peptide comprising at least part of the amino acid sequence RDSHPQAMQWNSTRFHQAL (SEQ ID NO: 2). A preferred specific fragment is MQWN (SEQ ID NO: 5).
Another preferred embodiment of a fragment is a peptide comprising at least part of the amino acid sequence SHPQAMQWNSTTFHQALLDPR. (SEQ ID NO: 3) A preferred specific fragment is STFHQA (SEQ ID NO: 6).
Another preferred embodiment of a fragment is a peptide comprising at least part of the amino acid sequence ALLDPRVRGLYFPAGGSSSGT (SEQ ID. NO: 4). A preferred specific fragment is VRGLYFPA (SEQ ID NO: 7).
Definition of optimal peptides (synthetic or recombinant) representing immunodominant domains of HBsAg, capable of replacing the existing peptides or proteins in diagnostic tests is of crucial importance in order to detect all possible HBsAg positive samples.
These peptides can be synthesized highly reproducible and are easily purified, thus well suited for further improvement and standardization of HBV-serology.
Synthetic peptides have the advantage of being chemically well defined, thus allowing easy and reproducible production at high yields, well suited for application in diagnostic assays which can be manufactured and used with greater reproducibility.
In contrast to natural HBsAg proteins, the peptides according to the present invention have the great advantage that these are of a safe non-infectious origin.
The term xe2x80x9cpeptidexe2x80x9d as used herein refers to a molecular chain of amino acids with a biological activity, and does not refer to a specific length of the product. Thus inter alia, proteins, fusion-proteins or -peptides oligopeptides. and polypeptides are included.
If required peptides according to the invention can be modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation. Functional variants like, for example, acid addition salts, amides, esters, and specifically C-terminal esters, and N-acyl derivatives of the peptides according to the invention are therefore also considered part of the present invention.
The term xe2x80x9cat least a part ofxe2x80x9d as used herein means a subsequence of the identified amino acid sequence. Said part or fragment is a region having one or more antigenic determinants of the HBsAg proteins. Fragments can inter alia be produced by enzymatic cleavage of precursor molecules, using restriction endonucleases for the DNA and proteases for the (poly)peptides. Other methods include chemical synthesis of the fragments or the expression of peptide fragments by DNA fragments.
The preparation of the peptides or fragments thereof according to the invention is effected by means of one of the known organic chemical methods for peptide synthesis or with the aid of recombinant DNA techniques which are also known in the art. Peptides according to the present invention can also be combined in a single molecule.
As already indicated above, the peptides according to the invention can likewise be prepared with the aid of recombinant DNA techniques. This possibility is of importance particularly when the peptide is incorporated in a repeating sequence (xe2x80x9cin tandemxe2x80x9d) or when the peptide can be prepared as a constituent of a (much larger) protein or polypeptide or as a fusion protein with, for example, (part of) xcex2-galactosidase. This type of peptides therefore likewise falls within the scope of the invention.
The peptides or fragments thereof prepared and described above can also be used to produce antibodies, both polyclonal and monoclonal and other binding molecules.
Methods of diagnosis in accordance with the invention can be carried out in vitro. According to the fourth aspect of the present invention, there is provided a method for the diagnosis of hepatitis B, the method comprising contacting the sample suspected to contain hepatitis B particles or antigens with the specific binding molecule as described above.
A preferred embodiment of the present invention is directed to a method for the diagnosis of hepatitis B, the method comprising contacting the sample suspected to contain hepatitis B particles or antigens with at least one specific binding molecule directed to the S-region of HBV and at least one specific binding molecule according to the present invention.
A particularly suitable method for the detection of HBsAg in a sample is based on a competition reaction between a peptide according to the invention provided with a labeling substance and hepatitis B particles or antigens (present in the sample) whereby the peptide and the antigen are competing with the specific binding molecule according to the present invention attached to a solid support.
Another preferred embodiment of the present invention is directed to a method for the detection of antibodies to hepatitis B, the method comprising contacting the sample suspected to contain antibodies to hepatitis B particles or antigens with at least one peptide according to the present invention.
In in vitro assays, some form of supports are used to immobilise the binding molecules or peptides according to the present invention. Supports which can be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an aldehyde particle (such as a ceramic magnetizable particle with active aldehyde surface groups), an erythrocyte, a dye sol, a metal sol or metal compound as sol particle, a carrier protein such as BSA or KLH.
Also some form of labeling is used to detect the antigen-antibody interaction. Labeling substances may be radioactive or non-radioactive such as a radioactive isotope, a fluorescent compound, a chemiluminescent compound, an enzyme, a dye sol, metal sol or metal compound as sol particle. Depending upon the format of the assay, either the specific binding molecules within the scope of the invention can be labeled, or other specific binding molecules, which bind to them are labeled. Immunoassays (including radioimmunoassays) and immunometric assays (including immunometric radloassays and enzyme-linked immunosorbent assays) can be used, as can immunoblotting techniques.
In vitro assays may take many formats. Some depend upon the use of labeled specific binding molecules such as antibodies (whose use is included within the scope of the invention), whereas some detect the interaction of antibody (or other specific binding molecule) and antigen by observing the resulting precipitation. These are well known in the art.
In vitro assays will often be conducted using kits. According to the fifth aspect of the present invention, there is provided an assay kit for the detection of a hepatitis B particle or antigen, the kit comprising a specific binding molecule as described above and means for detecting whether the specific binding molecule is bound to a hepatitis B particle or antigen.
The assay methodology may for example be any of the assays referred to above. Competitive and, especially, sandwich immunoassay kits are preferred. The specific binding molecule and the detection means may be provided in separate compartments of the kit. The specific binding molecule may be provided bound to a solid support. The detections means may comprise a detectable labeled second specific binding molecule (which itself may be an antibody (monoclonal but preferably polyclonal), which bind to the bound hepatitis B particle or antigen.
A preferred embodiment of the present invention to an assay kit for the detection of a hepatitis B particle or antigen, the kit comprising at least one binding molecule directed to the S-region of HBV and at least one specific binding molecule according to the present invention and means for detecting whether the specific binding molecule is bound to a hepatitis B particle or antigen.
Another preferred embodiment of the present invention to an assay kit for the detection of antibodies to hepatitis B, the kit comprising at least one peptide according to the present invention and means for detecting whether the peptide is bound to antibodies to hepatitis B.
Carrying out a sandwich reaction, for the detection of antibodies to hepatitis B the test kit may comprise, for example, a peptide according to the invention coated to a solid support, for example the inner wall of a microtest well, and either a labeled peptide according to the invention or a labeled anti-antibody.
For carrying out a competition reaction, the test kit may comprise a peptide according to the invention coated to a solid support, and a labeled specific binding molecule according to the invention.
According to the sixth aspect of the present invention, there is provided the use of a specific binding molecule according to the present invention for the in vitro diagnosis of hepatitis B. The antibodies and other binding molecules according to the present invention can be useful in the development and quality control of serologic assays and for the direct detection of HBV.
Also the use of the specific binding molecules according to the present invention in immunological and biochemical methods aiming to detect the full length protein in a test fluid or tissue specimen is provided.
The invention is further exemplified by the following examples: